Periodic Reporting for period 1 - CTL (Characterization of the molecular mechanism behind CTL - the major choline uptake system in eukaryotes)
Periodo di rendicontazione: 2020-05-01 al 2022-04-30
Choline is an essential nutrient for the human body with a critical importance for the fetal development of the brain, the nervous system and metabolism. It is a precursor in the synthesis of membrane phospholipids, the neurotransmitter acetylcholine and the methyl donor betaine. In order to be available for the human body it needs to be transported through the plasma membrane by specialized proteins, called membrane transporters. Transmembrane uptake of choline is the essential, but still enigmatic, first step in choline metabolism in the cell. Furthermore, uptake and cellular homeostasis of choline is affected in several diseases (e.g. Alzheimer, cardiovascular disease, fetal alcohol syndrome), brain ischemic events, aging and cancer; underlining its importance for human health.
The choline transporter-like (CTL) family forms a unique class of membrane transporters with five family members, that facilitate the majority of choline uptake in all eukaryotes. With no direct homologies to other transporter families, protein structures, especially with bound substrate, are of utmost importance to understand how this enigmatic transporter class facilitates substrate transport. Therefore, the overall objectives of this project were the structural, functional and biochemical characterization of CTL proteins.
The choline transporter-like (CTL) family forms a unique class of membrane transporters with five family members, that facilitate the majority of choline uptake in all eukaryotes. With no direct homologies to other transporter families, protein structures, especially with bound substrate, are of utmost importance to understand how this enigmatic transporter class facilitates substrate transport. Therefore, the overall objectives of this project were the structural, functional and biochemical characterization of CTL proteins.
During the funding period I worked extensively on proteins of the CTL family, towards their structural and functional characterization. I was able to establish the recombinant expression of several CTL proteins. I solubilized CTL proteins using different detergents from their membrane environment and purified these using chromatography methods (e.g. affinity chromatography, size exclusion chromatography).
Further sample analysis was conducted using biochemical and structural methods. Uptake assays for instance, were performed using xenopus oocytes expressing CTL proteins. Radioactive compounds were presented to the oocytes and uptake was measured by quantifying the radioactivity that was accumulated inside the oocytes.
Further sample analysis was conducted using biochemical and structural methods. Uptake assays for instance, were performed using xenopus oocytes expressing CTL proteins. Radioactive compounds were presented to the oocytes and uptake was measured by quantifying the radioactivity that was accumulated inside the oocytes.
We have not published our results yet, but we are currently finalizing the first paper on our finding on CTL proteins and we expect to publish another paper, that will deepen our knowledge on the choline transporter-like family.